Objectives:
Principle:Antibodies pervade almost every tissue and fluid in our body. Functionally antibodies can be characterized by their ability to bind both to antigens and to specialized cells or proteins of the immune system. Structurally antibodies are composed of one or more copies
of a characteristic unit that can be visualized as forming a Y-shape. Each Y contains four polypeptides. Two identical copies of a polypeptide called the light chain. Antibodies are divided into five classes – IgG, IgM, IgA, IgE and IgD on the basis of the number of Y-like units and the type of heavy chain polypeptide they contain. Show
IgG AntibodiesThe important structural features of antibodies are easiest to understand by considering IgG antibodies which contain
only one structural unit and also most abundant in serum. About 75% of all immunoglobulins in our body are IgG. Compared to the other classes of immunoglobulin it is small and light weighing in at about 146 kilodaltons. Its heavy chains are called gamma heavy chains and contain very little carbohydrate (about 3%).
IgG molecules have three protein domains. Two of the domains are identical and form the arms of the Y. Each arm contains a site that can bind to an antigen making IgG molecules bivalent. The third domain forms the base of the Y and this region is important in certain aspects of the immune response. The three domains may be separated from each other by cleavage with protease Papain. The two domains that carry the antigen binding sites are known as F(ab) fragments (named for the fragment having the antigen binding site) and the protein domain that is involved in immune regulation is termed as Fc fragment (for the fragment that crystallizes). The region between the F(ab)and Fc fragments is called the hinge. This segment allows lateral and rotational movement of the two antigen binding domains. The two heavy chain polypeptides in the Y structure are identical and are approximately 55000 daltons.The two light chains are also identical and are about 25000 daltons. One light chain associates with the anmino terminal region of one
heavy chain to form an antigen binding domain.The carboxy terminal region of the two heavy chains fold together to make the Fc domain. The two polypeptide chains are held together by disulfide bridges and non-covalent bonds. Cleavage of IgG using Proteolytic EnzymesThe structural aspects of antibodies using Proteolytic enzymes such as Papain and pepsin were extensively studied by porter (1959) and Nisonoff.et.al (1960). The protein specific enzymes break the
covalent peptide bond between two amino acid residues in the polypeptide chain, each enzyme having a special preference for a particular residue or residues. Enzymes commonly used are Papain isolated from the latex of the papaya tree “Carcia papaya“ and pepsin isolated from stomach juice. Papain splits the immunoglobulin molecule into three pieces of equal size. Thus two of these pieces are identical and are able to bind antigen, the third piece is different and is not
capable of antigen binding. The former is the Fab pieces (Fab- Fragment antigen binding) and later is Fc piece (Fc-Fragment crystalline) because it can be crystallized from a solution as a homogenous substance, Fab prepared from a pool of serum IgG molecules cannot be crystallized because they are such a heterogeneous bunch. The fragments obtained by the Proteolytic cleavage of antibodies can be further subjected to chemical treatment yields smaller fragments. For example the
treatment of Fab fragment with 2-Mercaptoethanol yields two smaller fragments one which is the L-chain while the other slightly larger than the L-chain. Also the prolonged treatment of Fc fragment with Papain cleaves this fragment further to produce Fc. By special chemical treatment it is also able to produce Fv fragments which consists of the VH and VL domains held together by noncovalent bonds.
Papain is a cysteine protease of the peptidase C1 family. Papain consists of a single polypeptide chain with three disulfide bridges and a sulfhydryl group necessary for activity of the enzyme. Papain cleaves immunoglobulin G molecules in the hinge reason which results in the generation of three ~50kDa fragments; two Fab domains and a Fc domain. The Papain-digested antibody is unable to promote agglutination, precipitation, opsonization, and lysis. Physical Properties and KineticsMolecular weight: 23,406 Da (amino acid sequence) Spectral properties:λmax: 278 nm 19 Solubility and Solution StabilityPapain is soluble in water at 10 mg/ml. The enzyme is typically diluted in buffer containing ~5 mM L-cysteine immediately before its usage. Activation/stabilizing agents include EDTA, cysteine, and dimercaptopropanol. Although Papain solutions have good temperature stability, the solution stability is pH dependent. Papain solutions are unstable under acidic conditions, i.e., at pH values
below 2.8, there is a significant loss in activity. A common inactive form of Papain obtained during isolation is a mixed disulfide formed between the active site sulfhydryl group of the protein and free cysteine. Papain solutions are stable to several denaturing agents, i.e., full activity is maintained after recrystallization in 70% methanol and in 8 M urea solutions. However, there is a significant loss in activity when Papain is exposed to 10% trichloroacetic acid or to 6 M
guanidine hydrochloride. SpecificityPapain will digest most protein substrates more extensively than the pancreatic proteases. Papain exhibits broad specificity, cleaving peptide bonds of basic amino acids, leucine, or glycine. It also hydrolyzes esters and amides. Papain exhibits a preference for an amino acid bearing a large hydrophobic side chain at the P2 position. It does not accept Val at the P1 position. Applications of PapainPapain is commonly used in cell isolation procedures where it has proven more efficient and less destructive than other proteases on certain tissues.
Why antibodies are fragmented?Proteolytic cleavage of antibodies results in defined antibody fragments. The analysis of antibody fragments offers several advantages over intact antibodies. Some of the advantages of antibody fragments are:
Note:Please check "Procedure tab" if you have issues loading the simulator.What are the effects of papain and pepsin on the antibody molecule?Immunoglobulin's are proteins and are generally resistant to digestion by enzymes. However, enzyme papain and pepsin cleave the immunoglobulin molecules. The fragments generated by papain and pepsin are useful for studies on immunoglobulin structure and functions.
What does pepsin do to antibodies?Pepsin cleaves the antibody to produce divalent F(ab')2 fragment and small peptides of Fc fragment by cleaving the heavy chains near the hinge regions. The resulting F(ab')2 fragment is comprised of two Fab units joined by disulfide bonds. The light chains remain intact and attached to the heavy chain.
What will happen if you digest antibody molecule with papain enzyme?Papain digestion: Fab from IgG
When IgG molecules are incubated with papain in the presence of a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragment and one Fc fragment (1).
What are the products when IgG antibodies are digested with pepsin and papain?Digestion of an IgG with papain produces three products: one Fc fragment and two identical Fab fragments. The Fab fragment is a 50 kDa disulfide bonded antibody fragment that retains its antigen binding activity but lacks the Fc portion of the IgG. The Fc fragment is composed of parts of two heavy chains.
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